Understanding receptor clustering via multivalent ligand display

Background

Extracellular signalling allows cells to communicate and respond to their environment. Ligand binding by cell surface receptors triggers downstream intracellular processes that ultimately dictate cell fate. Critically, these signalling events are concentration dependent with receptor clustering and oligomerisation being correlated to increased signal potency and persistence. However, the ligand organisation/valency necessary for activation is poorly understood. In this project, we will therefore create a modular polymer-protein array platform able to present precisely defined multi-ligand architectures to cells, enabling us to correlate downstream cell fate to receptor orientation and oligomerisation for the first time.

Objectives

To allow us to achieve this goal, we have three key objectives: i) To develop novel chemistries that can selectively target the N-termini of proteins for modification; ii) To synthesise precisely defined polymer-protein conjugate arrays with common cell signalling proteins; and iii) To correlate polymer-protein conjugate architecture to intracellular signalling activation and persistence.

Impact

While the importance of dimerisation or higher order clustering of receptors has been widely demonstrated, our mechanistic understanding is mostly limited to demonstrations of increased activation over single isolated receptor-ligand motifs - the optimal orientation, architecture, and valency of ligand presentation necessary for downstream signalling is poorly understood. The project described here directly addresses this challenge by providing a precise means to probe protein-ligand display, and thus study one of the key 'rules of life'. We will therefore enable unprecedented understanding of receptor-ligand interactions by providing insight into the concentration dependent nature of receptor clustering. Importantly, abnormal receptor activation is widely implicated in disease, and there is an urgent need for new tools for studying receptor clustering. High-purity growth factor preparations and low volume polymer arrays have only recently emerged while N-terminal protein targeting remains in its infancy. The novel combination of technologies described here is therefore timely.

By providing a facile means to conjugate any protein of interest to a polymer array library in a controlled manner, we will also deliver a powerful platform for high throughput multi-valent ligand screening. The output of this project will therefore create additional opportunities to 'integrate bioscience with innovation in the engineering and physical sciences', as described by the 'Transformative technologies' priority area of the BBSRC.